Animal protein-free media for cultivation of cells

ABSTRACT

The present invention relates to animal protein-free cell culture media comprising polyamines and a plant- and/or yeast-derived hydrolysate. The invention also relates to animal protein-free culturing processes, wherein cells can be cultivated, propagated and passaged without adding supplementary animal proteins in the culture medium. These processes are useful in cultivating cells, such as recombinant cells or cells infected with a virus, and for producing biological products by cell culture processes.

FIELD OF THE INVENTION

The present invention relates to animal protein-free cell culture mediacomprising a polyamine and a plant- and/or yeast-derived hydrolysate.The invention also relates to animal protein-free culturing processes,wherein cells can be cultivated, propagated and passaged without addingsupplementary animal proteins in the culture medium. These processes areuseful in cultivating cells, such as recombinant cells or cells infectedwith a virus, and for producing biological products by cell cultureprocesses.

BACKGROUND OF THE INVENTION

For cultivation of cells, particularly eukaryotic cells, and morespecifically mammalian cells, there is a constant need to use specialculture media that make available the growth nutrient substances thatare required for efficient growth of the cells and for the production ofthe proteins or viruses that are desired. For the efficient productionof biological products, such as viruses or recombinant proteins, it isimportant that an optimal cell density is achieved as well as theprotein expression itself is increased to obtain maximal product yield.

Cell culture media formulations have been supplemented with a range ofadditives, including undefined components like fetal calf serum (FCS),several animal derived proteins and/or protein hydrolysates of bovineorigin.

In general, serum or serum-derived substances, such as albumin,transferrin or insulin, may contain unwanted agents that can contaminatethe cell cultures and the biological products obtained therefrom.Furthermore, human serum derived additives have to be tested for allknown viruses, including hepatitis and HIV, that can be transmitted byserum. Moreover, bovine serum and products derived therefrom bear therisk of BSE contamination. In addition, all serum-derived products canbe contaminated by unknown constituents. In the case of serum or proteinadditives that are derived from human or other animal sources in cellculture, there are numerous problems (e.g. the varying quality incomposition of the different batches and the risk of contamination withmycoplasma, viruses or BSE), particularly if the cells are used forproduction of drugs or vaccines for human administration.

Therefore, many attempts have been made to provide efficient hostsystems and cultivation conditions, which do not require serum or otheranimal protein compounds. Simple serum free medium typically includesbasal medium, vitamins, amino acids organic or inorganic salts, andoptionally additional components to make the medium nutritionallycomplex.

Soy hydrolysates are known to be useful for fermentation processes andcan enhance the growth of many fastidious organisms, yeasts and fungi.WO 96/26266 describes that papaic digests of soy meal are a source ofcarbohydrate and nitrogen and many of the components can be used intissue culture. Franek et al. (Biotechnology Progress (2000) 16,688-692) describe growth and productivity promoting effects of definedsoy hydrolysate peptide fractions.

WO 96/15231 discloses serum-free medium composed of the syntheticminimal essential medium and yeast extract for propagation of vertebratecells and virus production process. A medium formulation composed of abasal cell culture medium comprising a rice peptide and an extract ofyeast and enzymatic digest thereof, and/or a plant lipid for growth ofanimal cells is disclosed in WO 98/15614. A medium comprising purifiedsoy hydrolysate for the cultivation of recombinant cells is disclosed inWO 01/23527. WO 00/03000 discloses a medium that comprises a soyhydrolysate and a yeast extract, but also requires the presence ofrecombinant forms of animal proteins, such as growth factors.

EP-A-0 481 791 describes a biochemically defined culture medium forculturing engineered CHO cells, which is free from protein, lipid andcarbohydrate isolated from an animal source, further comprising arecombinant insulin or insulin analogue, 1% to 0.025% w/v papaindigested soy peptone and putrescine. WO 98/08934 describes a serum-freeeukaryotic cell culture comprising hydrolyzed soy peptides (1-1000mg/L), 0.01 to 1 mg/L putrescine and a variety of animal-derivedcomponents, including albumin, fetuin, various hormones and otherproteins. In this context, it should be also noted that putrescine isalso known to be contained in standard media like DMEM/Ham's F12 in aconcentration of 0.08 mg/L.

However, the media known in the state of art are often nutritionallyinsufficient and/or must be supplemented with animal-derived proteinsupplements or recombinant versions of proteins, such as insulin,insulin like growth factor or other growth factors.

Therefore, a current need exists to increase the yield of expressedrecombinant protein or any other expression product, and specific growthrate of cells, and to provide an optimal cell culture medium completelyfree of animal proteins for production of biological products, such asthose used as pharmaceuticals or vaccines in humans.

On the basis of soy peptone extracts (also designated as “soyhydrolysates”) media have been developed, which do not contain animalproteins. However, the quality of commercially available lots of soyhydrolysates varies extremely and as a result, there are largevariations in the production of recombinant proteins or viral products(a variation of up to a factor of 3) as a function of the lots of soyhydrolysates used (“lot-to-lot variation”). This draw back affects theproliferation of the cells as well as the protein expression of eachcell.

Therefore, there is a need for an animal protein-free cell culturemedium which is completely free of animal proteins and overcomes atleast one of the above-mentioned problems.

SUMMARY OF THE INVENTION

An object of the present invention is to provide an animal protein-freecell culture medium which does not contain any added supplementaryproteins derived from an animal source and/or recombinant animalproteins, which allows efficient cell growth and in particular proteinproduction in a continuous quality with respect to the amount ofexpression per cell. A further object of the present invention is toprovide a method for cultivating cells and a method for efficientexpression of recombinant proteins which are free of animal proteins.

Another object of the present invention is to reduce plant and/or yeastderived hydrolysate in order to overcome inhibitory effects which wouldnegatively impact the production yield of a desired recombinant or viralproduct. Hydrolysates were surprisingly found to be the cause of thelot-to-lot variations in production.

The animal protein-free cell culture medium according to the inventioncomprises at least one polyamine and a plant- and/or yeast-derivedhydrolysate, wherein the polyamine preferably originates from a sourceother than the protein hydrolysate.

Surprisingly, the addition of at least one polyamine, in particular theaddition of putrescine, to the animal protein-free cell culture mediumprovides the advantageous effect not only to promote the cell growth butin particular to increase the protein expression per cell and, inparticular, recombinant protein expression per cell.

Further, the animal protein-free medium according to the presentinvention allows consistent cell growth and increased yield of desiredproducts, particularly of target proteins such as recombinant proteins,independent of the quality or lot variations of the protein hydrolysate,in particular of the vegetable hydrolysates, in the animal protein-freecell culture medium. The specific supplementation of cell culture mediawith polyamines and a plant- and/or yeast-derived hydrolysate actssynergistically to increase cell growth, cell specific productivity andfinal cell density.

Therefore, the animal protein free medium according to the presentinvention is more favorable for recombinant protein expression and cellgrowth rate compared to the media known in the art. Furthermore, theanimal protein-free medium according to the present invention allows thereduction of the amount of protein hydrolysate to be added to a givenvolume of the cell culture medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph which compares (A) the volumetric FVIII-CoAproductivity (expressed in [U/L/D]=FVIII COA Units per L reactor volumeper day and (B) the specific growth rate (μ expressed in [d⁻¹]=1 perday) of GD8/6 cells as a function of the media used for culture, whichwere supplemented with different lots (K119-1, K138-1, M022963, M024423,M022453) of soy hydrolysates (0.4% (w/v)).

FIG. 2 shows a table which compares the volumetric FVIII-CoAproductivity of GD8/6 cells grown in media with different soyhydrolysate concentrations.

FIG. 3 shows a graph which compares the volumetric FVIII-CoAproductivity of GD8/6 cells as a function of the media used for culture,which were supplemented with 5 different lots (K119-1, K138-1, M022963,M024423, M022453) of soy hydrolysates (0.25% (w/v)) (A) in the absenceof putrescine and (B) in the presence of 1 mg/L putrescine.2HCl.

FIG. 4 shows a graph which compares the specific growth rates of GD8/6cells as a function of the media used for culture, which weresupplemented with 5 different lots (K119-1, K138-1, M022963, M024423,M022453) of soy hydrolysates (0.25% (w/v)) (A) in the absence ofputrescine and (B) in the presence of 1 mg/L putrescine.2HCl.

FIG. 5 shows a table which compares the volumetric FVIII-CoAproductivity (QP [U/L/D]) and the specific growth rate (μ[d−1]) of GD8/6cells and their standard deviation grown in media with 5 differentselected lots (K119-1, K138-1, M022963, M024423, M022453) of soyhydrolysates (0.4% (w/v) or 0.25% (w/v)) with the same soy hydrolysates(0.25% (w/v)) with and without putrescine.2HCl at 1 mg/L.

FIG. 6 shows a table which describes the average putrescineconcentrations found in soy hydrolysates (0.4% (w/v) in cell culturemedium) from different manufacturers.

FIG. 7 shows a table which compares the effect of soy hydrolysate (0.4%(w/v)) and soy hydrolysate (0.25% (w/v))+1,8 mg/L putrescine.2HCl on thevolumetric productivity (QP expressed in [mg IgG1/L reactor volume/day]and cell specific productivity (qp [μg IgG1/10E06 Cells/d]) in ARH77cells secreting a monoclonal antibody.

FIG. 8 shows a graph which compares the effect of soy hydrolysate (0.25%(w/v)) and soy hydrolysate (0.25% (w/v))+1 mg/L putrescine (1,8 mg/Lputrescine.2HCl) on the cell specific erythropoeitin (EPO)-productivityof recombinant BHK cells (EPO production (Units)/glucose consumption(g).

FIG. 9 shows a table comparing the effect of putrescine, ornithine andspermine over a wider concentration range (0-18 mg/L) on the specificgrowth (μ absolute, μ relative) and the cell specific productivity (Qpabsolute, Qp relative) of GD8/6 cells cultivated in BAV-mediumcontaining 0,0% soy hydrolysate and no amines, or BAV-medium containinga reduced soy hydrolysate concentration of 0,25% supplemented withpolyamines in the concentration range indicated above. BAV−SP 0.25%=BAVmedium containing 0.25% soy hydrolysate; BAV−SP 0.4%=BAV mediumcontaining 0.4% soy hydrolysate; BAV w/o soy no polyamines=BAV mediumcontaining neither soy hydrolysate nor polyamines.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention relates to an animal protein-free cellculture medium comprising at least one polyamine and a plant- and/oryeast-derived hydrolysate, in a concentration sufficiently reduced inorder to avoid potential inhibitory effects of the hydrolysate.

The term “polyamine” refers to any of a group of organic compoundscomposed of carbon, nitrogen, and hydrogen, and containing two or moreamino groups. For example, the term encompasses molecules selected fromthe group consisting of cadaverine, putrescine, spermidine, spermine,agmatine, and ornithine.

Unless stated differently, concentration values indicated throughoutthis document refer to the free base form of the component(s).

In a preferred embodiment of the animal protein-free cell culture mediumthe concentration of the polyamine is present in a concentration rangingfrom about 0.5 mg/L to about 30 mg/L, more preferably from about 0.5mg/L to about 20 mg/L, even more preferably from about 0.5 mg/L to about10 mg/L, more preferably from about 2 mg/L to about 8 mg/L, mostpreferably from about 2 to about 5 mg/L in the medium.

In a preferred embodiment the total concentration of the plant- and/oryeast-derived protein hydrolysate in the animal protein-free cellculture medium is about 0.05% to about 5% (w/v), more preferably about0.05% to about 2% (w/v), more preferably about 0.05% to about 1% (w/v),more preferably about 0.05% to about 0.5% (w/v), most preferably about0.05% to about 0.25% (w/v); i.e. if the medium contains a plant- and ayeast derived protein hydrolysate, the total concentration is calculatedby the summing up the concentration values of each of the proteinhydrolysate components contained in the medium.

The term “animal protein free cell culture medium” according to theinvention refers to a medium that does not contain proteins and/orprotein components from higher multicellular non-plant eukaryotes.Typical proteins that are avoided are those found in serum andserum-derived substances, such as albumin, transferrin, insulin andother growth factors. The animal protein free cell culture medium isalso free of any purified animal derived products and recombinant animalderived products as well as protein digests and extracts thereof orlipid extracts or purified components thereof. Animal proteins andprotein components are to be distinguished from non-animal proteins,small peptides and oligopeptides obtainable from plants (usually 10-30amino acids in length), such as soy bean, and lower eukaryotes, such asyeast which may be included into the animal protein free cell culturemedium according to the invention.

The animal protein free culture medium according to the invention may bebased on any basal medium such as DMEM, Ham's F12, Medium 199, McCoy orRPMI generally known to the skilled worker. The basal medium maycomprise a number of ingredients, including amino acids, vitamins,organic and inorganic salts, and sources of carbohydrate, eachingredient being present in an amount which supports the cultivation ofa cell which is generally known to the person skilled in the art. Themedium may contain auxiliary substances, such as buffer substances likesodium bicarbonate, antioxydants, stabilisers to counteract mechanicalstress, or protease inhibitors. If required, a non-ionic surfactant suchas mixtures of polyethylene glycols and polypropylene glycols (e.g.Pluronic F68®, SERVA) can be added as a defoaming agent.

The polyamine employed for the animal protein free culture mediumaccording to the invention may be selected from the group consisting ofcadaverine, putrescine, spermidine, spermine, agmatine, ornithine, andcombinations thereof. Most preferably, the animal protein free culturemedium contains ornithine, putrescine and spermine.

In an preferred embodiment of the animal protein free culture medium thepolyamine controls DNA- and RNA-synthesis, cell proliferation, celldifferentiation, membrane stabilization, and/or antioxidativeDNA-protection. Putrescine, spermidine, spermine, and ornithine areexamples of polyamines which exhibit these functions. Another example ofa polyamine is cadaverine.

In another preferred embodiment of the animal protein-free cell culturemedium according to the invention the polyamine originates from a sourceother than the protein hydrolysate.

In a further preferred embodiment of the animal protein-free cellculture medium the polyamine is present in a concentration ranging fromabout 0.5 to about 30 mg/L, more preferably from about 0.5 mg/L to about20 mg/L, even more preferably from about 0.5 mg/L to about 10 mg/L, morepreferably from about 2 mg/L to about 8 mg/L, most preferably from about2 to about 5 mg/L in the medium, and the plant- and/or yeast-derivedprotein hydrolysate is present in the medium in a concentration rangingfrom about 0.05% to about 5% (w/v), more preferably about 0.05% to about2% (w/v), more preferably about 0.05% to about 1% (w/v), more preferablyabout 0.05% to about 0.5% (w/v), most preferably about 0.05% to about0.25% (w/v).

The plant-derived protein hydrolysate used for the animal protein-freecell culture medium according to the invention is preferably selectedfrom the group consisting of a cereal hydrolysate and/or a soyhydrolysate. The soy hydrolysate may be a highly purified soyhydrolysate, a purified soy hydrolysate or crude soy hydrolysate.

The term “hydrolysate” includes any enzymatic digest of a vegetable oryeast extract. The “hydrolysate” can be further enzymatically digested,for example by papain, and/or formed by autolysis, thermolysis and/orplasmolysis. Hydrolysates to be used according to the present inventionare also commercially available, such as HyPep 1510®, Hy-Soy®, Hy-Yeast412® and Hi-Yeast 444®, from sources such as Quest International,Norwich, N.Y., OrganoTechnie, S.A. France, Deutsche Hefewerke GmbH,Germany, or DMV Intl. Delhi, N.Y. Sources of yeast extracts and soyhydrolysates are also disclosed in WO 98/15614, WO 00/03000, WO 01/23527and U.S. Pat. No. 5,741,705.

The hydrolysates are preferably purified from crude fraction, becauseimpurities could interfere with efficient cultivation. Purification canbe carried out by ultrafiltration or Sephadex chromatography, forexample with Sephadex 25 or Sephadex G10 or equivalent materials, ionexchange chromatography, affinity chromatography, size exclusionchromatography or reverse-phase-chromatography. The fractions maycontain hydrolysates of defined molecular weight, preferably up to about1000 Dalton, more preferably up to about 500 Dalton, most preferably upto about 350 Dalton. At least about 90% of the hydrolysate haspreferably a molecular weight of up to about 1000 Dalton. The averagemolecular weight of the hydrolysates lies preferably between about 220and about 375 Daltons. The pH value of the hydrolysate should be in therange of from about 6.5 to about 7.5. The total nitrogen content ispreferably between about 5 and about 15%, and the ash content ispreferably up to about 20%. The free amino acid content is preferablybetween about 5% and about 30%. The endotoxin content is preferably lessthan about 500 U/g.

The invention also provides a method of using at least one polyamine foraddition to an animal protein-free cell culture medium containing aplant- and/or yeast-derived protein hydrolysate, for increasing theprotein expression yield in the cultured cells. According to a preferredembodiment of the invention, the polyamine is present in the culturemedium in a total concentration ranging from about 0.5 to about 30 mg/L,more preferably from about 0.5 mg/L to about 20 mg/L, even morepreferably from about 0.5 mg/L to about 10 mg/L, more preferably fromabout 2 mg/L to about 8 mg/L, most preferably from about 2 to about 5mg/L in the medium. Preferably, the polyamine is selected from the groupconsisting of cadaverine, putrescine, spermidine, spermine, agmatine,ornithine, and combinations thereof. Preferably, the plant- and/oryeast-derived protein hydrolysate is present in the medium in aconcentration ranging from about 0.05% to about 5% (w/v), morepreferably about 0.05% to about 2% (w/v), more preferably about 0.05% toabout 1% (w/v), more preferably about 0.05% to about 0.5% (w/v), mostpreferably about 0.05% to about 0.25% (w/v).

The present invention further relates to a method for cultivating cells,comprising the steps of:

-   -   (a) providing an animal protein-free cell culture medium        according to the invention, and    -   (b) propagating the cells in the medium to form a cell culture.

In a preferred embodiment the animal protein-free cell culture mediumcomprises at least one polyamine and a plant- and/or yeast-derivedhydrolysate. Preferably the polyamine originates from a source otherthan the protein hydrolysate.

The present invention is not limited to any type of cells. In apreferred embodiment of the invention the cells used are for examplemammalian cells, insect cells, avian cells, bacterial cells, yeastcells. The cells may be for example stem cells or recombinant cellstransformed with a vector for recombinant gene expression, or cellstransfected with a virus for producing viral products. The cells mayalso be for example cells producing a protein of interest withoutrecombinant transformation, e.g. a B-cell producing an antibody, whichmay be transformed into an immortalized status e.g. by viral infectionlike Epstein Barr Virus infection. The cells may also be for exampleprimary cells, e.g. chicken embryo cells, or primary cell lines.Preferred are cells that are used for in vitro virus production. In apreferred embodiment the cells may be BSC cells, LLC-MK cells, CV-1cells, COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAFcells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells,MDOK cells, BHK-21 cells, CHO cells, NS-1 cells, MRC-5 cells, WI-38cells, BHK cells, 293 cells, RK cells, and chicken embryo cells.

The cells used according to the present invention may be cultivated by amethod selected from the group of batch-cultivation,feed-batch-cultivation, perfusion cultivation and chemostate-cultivationall of which are generally known in the field.

The present invention further relates to a method for expressing atarget protein such as a heterologous or autologous protein or arecombinant protein, comprising the steps of:

-   -   a) providing a culture of cells that have been grown in an        animal protein-free cell culture medium according to the        invention; and    -   b) introducing a nucleic acid sequence comprising a sequence        coding for the target protein into the cells;    -   c) selecting the cells carrying the nucleic acid sequence; and    -   d) selectively inducing the expression of the target protein in        the cells.

In a preferred embodiment the animal protein-free cell culture mediumcomprises at least one polyamine and a plant- and/or yeast-derivedhydrolysate. Preferably, the polyamine originates from a source otherthan the protein hydrolysate.

The nucleic acid sequence comprising a sequence coding for the targetprotein may be a vector. The vector may be a virus or a plasmid. Thesequence coding for a target protein may be a specific gene or abiological functional part thereof. In a preferred embodiment the targetprotein is at least a biologically active part of a blood coagulationfactor such as the Factor VIII or at least a biologically active part ofa protein involved in the production of red blood cells and angiogenesissuch as erythropoeitin, or a monoclonal antibody.

Preferably, the nucleic acid further comprises other sequences suitablefor controlled expression of a target protein such as promotorsequences, as enhancers, TATA boxes, transcription initiation sites,polylinkers, restriction sites, poly-A-sequences, protein processingsequences, selection markers, and the like which are generally known tothe person skilled in the art.

Most preferred are the following cell lines transformed with arecombinant vector for the expression of the respective products: CHOcells for the production of recombinant coagulation factor VIII, BHKcells for the production of recombinant erythropoietin, Epstein Barrvirus transformed, immortalized human B cells for the production ofhuman antibodies.

The present invention further relates to a method for producing a virusor part of a virus, comprising the steps of:

-   -   a) providing a culture of cells that have been grown in an        animal protein-free cell culture medium according to the        invention; and    -   b) infecting the cells with a virus;    -   c) selecting the virus-infected cells; and    -   d) incubating the cells to propagate the virus.

In a preferred embodiment the animal protein-free cell culture mediumcomprises at least one polyamine and a plant- and/or yeast-derivedhydrolysate. More preferably, the polyamine originates from a sourceother than the protein hydrolysate.

The virus used in the method according to the invention may be anypathogenic virus, mammalian, preferably human virus, such as a vacciniaor attenuated vaccinia virus, e.g. for smallpox vaccines, coronavirus,preferably SARS virus, e.g. for production of SARS vaccines,orthomyoxyvirus, preferably influenza virus, e.g. for production ofinfluenza vaccines, paramyxovirus, retrovirus, influenza A or B virus,Ross River virus, flavivirus, preferably West Nile virus or FSME virus(i.e. tick borne encephalitis virus), e.g. for the production of therespective vaccines, picornavirus, arena virus, herpesvirus, poxvirus oradenovirus.

The virus may be a wild-type-virus, an attenuated virus, a reassortantvirus, or a recombinant virus or combinations thereof, e.g. attenuatedand recombinant. In addition, instead of actual virions being used toinfect cells with a virus, an infectious nucleic acid clone may be used.Split virions may also be used.

The method for expressing a protein or producing a virus may be used forproducing immunogenic compositions comprising a virus or a virusantigen.

The cells used for the method for producing a virus may be selected fromthe group consisting of mammalian cells, insect cells, avian cells,bacterial cells, and yeast cells. Preferably, the cells are cultivatedby a method selected from the group consisting of batch-cultivation,feed-batch-cultivation, perfusion cultivation and chemostat-cultivation.

Preferred combinations of cells with viruses for producing a virus orpart of a virus are Vero cell/attenuated vaccinia, Vero cell/Vaccinia,Vero cell/Hepatitis A, Vero cell/Influenza Virus, Vero cell/West NileVirus, Vero cell/SARS Virus, chicken embryo cells/FSME virus.

The present invention further relates to a method of using the animalprotein-free cell culture medium according to the invention forculturing cells expressing a target protein.

The present invention will now be further illustrated in the followingexamples, without being limited thereto.

EXAMPLES Example 1 BAV-medium

Animal protein free medium was prepared with basal DMEM/HAM's F12 (1:1)medium supplemented with inorganic salts, amino acids, vitamins andother components (Life technologies, 32500 Powder). Also added wereL-glutamine (600 mg/L), ascorbic acid (20 μM), ethanol amine (25 μM),Synperonic® (SERVA) (0.25 g/L), sodium selenite (50 nM). Additionally,essential amino acids were supplemented to the cell culture medium.Further, varying concentrations of soy hydrolysate (Quest Technologies,NY or DMV Intl., NY ) in the range of 0,0-1,0% and varyingconcentrations of polyamines (0-10 mg/L) were added (FIG. 1-9)

Example 2

Cell cultures of recombinant mammalian cells (e.g. CHO-cells stablyexpressing Factor VIII=GD8/6-cells) were grown in suspension in achemostat culture in 10 l bioreactors. The culture conditions of 37° C.,oxygen saturation 20% and pH 7.0 to 7.1 were kept constant. The cultureswere supplied with a constant feed of BAV-medium as defined in Example 1additionally supplemented with soy hydrolysates in the range of 0,1-1,0%and/or addition of putrescine.2HCl in the range of 0-1 mg/L (cf. FIG.1-5).

Small scale experiments with GD8/6 cells in suspension culture werecarried in Techne spinner flasks at 200 ml working volume in batchrefeed mode at 37° C., without pH and pO2 control. The cultures weresupplied with BAV-medium as defined in Example 1 without supplementationof soy hydrolysate and polyamines, or supplemented with soy hydrolysatein the range of 0.1-0,4% and/or putrescine.2HCl, ornithine.HCl,spermine.4HCl in the range of 0-18 mg/L (equivalent to 0-10 mg/L of thepolyamine without .HCl (cf. FIG. 9).

Example 3 cf. FIG. 1 to 5 7, and 9

Cell counts from suspension cells or immobilized cells were determinedeither by counting with a CASY® cell counter as described by Schärfe etal., (Biotechnologie in LaborPraxis 10: 1096-1103 (1988)) or by citricacid extraction and flourescent staining of the nuclei followed bycounting with a NucleoCounter® (Chemometec, DK). The specific growthrate (μm) is calculated from the increase of the cell densities (X_(t))and/or the dilution rate (D) of the steady state of chemostat culturesof suspensions cells over a certain time interval (t):μ=D+In(Xt/X0)/t

Example 4

The activity of Factor VIII (FVIII) (cf. FIGS. 1 to 5 and 9) wasmeasured by a chromogenic assay (Chromogenic, Sweden). The activity oferythropoeitin (cf. FIG. 8) and the monoclonal antibody titer (cf. FIG.7) were measured by ELISA test systems.

The volumetric productivity is calculated from the amount of activityunits or antigen titers yielded per liter reactor volume per day (U/L/dor mg/L/d) in the respective production systems.

The cell specific productivity is defined as the specific amount ofproduced protein (U or μg) per number of cells per day (cf. FIGS. 7 and9) or as the specific amount of produced protein (U) produced per amountof D-glucose consumed by the cells (cf. FIG. 8).

Example 5

GD8/6 cells were supplied with BAV-medium containing 0.4% (w/v) ofdifferent soy hydrolysate lots. The volumetric FVIII-productivity variedfrom about 600 to 1800 U/L/d and the specific growth rates varied offrom 0.35 to 0.52 μ[d−1] between the different lots (cf. FIG. 1). Thisindicates that the soy hydrolysate lots at the 0,4% concentration doesnot allow consistent growth of the GD8/6 cells, possibly due toinhibitory substances affecting the specific growth rate (μ) which arecontained in the soy hydrolysates.

Example 6

GD8/6 cells were supplied with BAV-medium containing differentconcentrations of soy hydrolysate lot M022257 (in the range of 0.15-1,0%w/v). The volumetric FVIII-productivity varied of from 500 to 1.100U/L/d and reached an optimum productivity of 1.100 U/L/d at a soyhydrolysate concentration of 0.4% (w/v) (cf. FIG. 2).

Example 7

GD8/6 cells were supplied with BAV-medium containing 0.25% (w/v) of thesame 5 different soy hydrolysate lots as described in Example 5 (FIGS.3A and 4A) and 0.25% (w/v) soy hydrolysate of the same soy hydrolysatelots additionally supplemented with 1 mg/L putrescine.2 HCl (FIGS. 3Band 4B), respectively. The volumetric FVIII-productivity varied of from1700 U/L/d to 500 U/L/d in the cells grown BAV-SP medium containing0.25% (w/v) soy hydrolysate of different soy hydrolysate lots (FIG. 3A).The specific growth rate varied of from 0.58 to 0.24 μ[d−1], indicatingthat the reduction of the soy hydrolysate concentration does not lead toan improved or more consistent growth rate of the cells (FIG. 4A).

In contrast, only minor variations of the volumetric FVIII-productivity(FIG. 3B) and specific growth rates (FIG. 4B) between the same soyhydrolysate lots are observed in the cells grown BAV-medium containing0.25% (w/v) soy hydrolysate when supplemented with 1 mg/Lputrescine.2HCl. The addition of 1 mg/L putrescine.2HCl approximatelycompensates the reduction of this polyamine by the reduction of soyhydrolysate concentration from 0.4% (w/v) to 0.25% (w/v). From this itcan be concluded that not the concentration of the polyamine itself, butthe addition of the polyamine in combination with the reduction of thesoy hydrolysate concentrations leads to a reduction of inhibitorysubstances which reduce growth and productivity (see Example 5).Furthermore, the addition of putrescine also leads to an overproportional increased volumetric productivity of FVIII due to anincrease of the cell specific FVIII productivity (FIG. 5).

Thus addition of putrescine to animal protein-free cell culture medianot only promotes protein expression rate of cultured cells but it alsoreduces the amount of plant hydrolysate to be included into the culturemedia in order to obtain the same cell growth. As a result, culturemedia become less affected by the lot-by-lot variation of quality ofplant hydrolysate and thus an overall improvement of the cell cultureconditions is achieved.

Example 8

FIG. 5 comprises the statistical analysis of the Examples shown in FIG.1, 2 and 4: GD8/6 cells were supplied with BAV-medium containing 0.4%(w/v) of soy hydrolysate or 0.25% (w/v) soy hydrolysate or 0.25% (w/v)soy hydrolysate and 1 mg/L putrescine.2HCl. Standard deviations arecalculated based on five selected lots of soy hydrolysates (K119-1,K138-1, M022963, M024423, M022453). The volumetric and cell specificFVIII-productivity and the specific growth rate with 0.25% (w/v) soyhydrolysate was lower than with 0.4% (w/v) soy hydrolysate, whichconfirms the optimum depicted in FIG. 2. However, the volumetric andcell specific FVIII-productivity and the specific growth rate increasesin cell culture medium containing 0.25% (w/v) soy hydrolysate+1 mg/Lputrescine.2HCl. Further, the standard deviation calculated from fivedifferent lots of soy hydrolysates is significantly reduced (cf. FIG. 5[QP [U/L/D]=volumetric productivity; qp [mU/106 cells/day]=cell specificproductivity).

Example 9

Examples 7 and 8 show that putrescine is an active compound supportingcell growth and, more specifically protein expression. Therefore, theconcentration of putrescine from different soy hydrolysate lots from 2different suppliers (Quest and DMV) were quantitatively analysed by aHPLC method and evaluated statistically. The concentration in the cellculture media prepared with soy hydrolysate from both suppliers wasapproximately 2.3 mg/L putrescine, when soy hydrolysate was added to themedium in a concentration of 0.4% (w/v) (cf. FIG. 6).

Example 10

ARH77 cells (human lymphoblastoid cell line stably expressing hIgG) weregrown in a perfusion culture after immobilization on macroporousmicrocarriers in a 80 L stirred tank bioreactor at 37° C., pH 7,0-7,2and pO2 20-80% air saturation, supplied with BAV medium containing 0.4%(w/v) of soy hydrolysate or 0.25% (w/v) soy hydrolysate+1,8 mg/Lputrescine.2HCl. Arithmetic means and standard deviations werecalculated from data points representing the steady states for therespective medium formulations. The volumetric hIgG-volumetricproductivity/cell specific productivity in BAV-medium supplemented with0.4% (w/v) soy hydrolysate was lower than in BAV-medium supplementedwith 0.25% (w/v) soy hydrolysate+1,8 mg/L putrescine.2HCl. Thisexperiment indicates that the medium composition according to thepresent invention is capable to promote also the expression ofmonoclonal antibodies from a transformed cell line. Further, thespecific medium composition can also be used in perfusion cultures (cf.FIG. 7).

Example 11

Recombinant BHK cells were grown to confluence in 5% (v/v) fetal calfserum containing medium. The cells were washed with protein-free mediumand incubated for 3 days in BAV medium supplemented with 0.25% (w/v) soyhydrolysate or 0.25% (w/v) soy hydrolysate+1,8 mg/L putrescine.2HCl(FIG. 8). Since no cell counting in this experiment was performed, theglucose consumption rate (g/L) was measured over three days to proveequivalent biomass in the culture system. The EPO-activity (mU/ml) wascorrelated with the glucose consumption rate (g/L) over three days. Theaddition of putrescine gives a 16% increase in EPO productivity comparedto BAV-medium merely supplemented with 0.25% (w/v) soy peptone. Thisexperiment also indicates that the medium composition according to thepresent invention is capable to promote the expression of differentrecombinant proteins.

Example 12

To prove the specific effect of putrescine, ornithine and spermine overa wider concentration range (0-18 mg/L equivalent to 0-10 mg/L of thepolyamine without -.HCl) an experiment was carried where the GD8/6 cellswere incubated in Techne spinner flasks at 1-1,5 E06 cells/ml inBAV-medium containing 0,25% and 0,4% soy hydrolysate without polyamines,and BAV-medium containing the reduced soy hydrolysate concentration of0,25% with the polyamines in the above mentioned concentration range.All three polyamines in the investigated concentration range resulted ina significant increase of cell specific productivity (expressed inmU/106 cells/day) compared to the unsupplemented medium formulation with0,25% soy hydrolysate, or the increased concentration of 0,4%. Theincrease of the cell specific productivity is clearly not correlatingwith an increased specific growth rate, which confirms the specificeffect on the expression rate of recombinant FVIII of the GD8/6 cells(FIG. 9).

1. A method for producing a virus, comprising the steps of: (a)providing a culture of cells that have been grown in an animalprotein-free cell culture medium comprising at least one polyamine andat least one protein hydrolysate derived from the group consisting ofplants and yeast, and (b) infecting the cells with the virus; (c)selecting the virus-infected cells; and (d) incubating the cells topropagate the virus.
 2. The method according to claim 1, wherein thecells are selected from the group consisting of mammalian cells, insectcells, avian cells, bacterial cells, and yeast cells.
 3. The methodaccording to claim 1, wherein the cell/virus combination is selectedfrom the group consisting of Vero cell/attenuated vaccinia, Verocell/vaccinia, Vero cell/hepatitis A, Vero cell/influenza virus, Verocell/West Nile virus, Vero cell/SARS virus, and chicken embryocells/FSME virus.
 4. The method according to claim 1 wherein the cellsare cultivated by a method selected from the group consisting ofbatch-cultivation, feed-batch-cultivation, perfusion cultivation, andchemostat-cultivation.